The objectives of this research are to elucidate the mechanism of chronic (long-term) tropic hormonal regulation of steroidogenic enzyme synthesis and activity in tumors utilizing rat and mouse Leydig tumor cells in monolayer culture. We have determined that cholesterol synthesized de novo under appropriate conditions may be the major source of cholesterol utilized for steroidogenesis in the tumor cells. We are determining changes in the content of the three components of the cholesterol side chain cleavage enzyme, i.e., cytochrome P450scc, testodoxin, and testodoxin reductase after hCG treatment. The proteins of the Leydig tumor cells are radiolabeled with [35S]-methionine. The extent of radiolabeling of the three proteins of the cholesterol, side-chain cleavage enzyme as well as sterol-carrier protein are determined after varying times of hCG treatment by use of immunoabsorbent techniques and SDS polyacrylamide gel electrophoresis (SDS-PAGE). These immunoisolations rely on the ability of IgG fractions of antisera against purified bovine adrenal and rat liver proteins to cross-react with the corresponding rat Leydig tumor cell proteins. We shall determine which serum or growth factor may serve as a putative second tropic agent for Leydig tumor cells by promoting cell growth and synthesis of steroidogenic enzymes and proteins. Cell cultures will be radiolabeled with [32P]-Pi in the presence or absence of hCG. We shall use IgG fractions of antisera against bovine adrenal cytochrome P450scc, adrenodoxin, adrenodoxin reductase, and rat liver sterol carrier protein to immunoisolate specifically the corresponding proteins from Leydig tumor cell lysates. After SDS-PAGE and visualization with autoradiography, the influence of gonadotropin on the extent of phosphorylation of these proteins will be assessed. Total RNA will be isolated from cells cultured in the presence or absence of hCG for varying periods and will be translated in a cell-free translation system. After immunoisolation, SDS-PAGE, and autoradiography we shall determine the role of hCG to alter the content of mRNA that directs the in vitro synthesis of the cholesterol, side-chain cleavage enzyme proteins, and sterol carrier protein. (D)